Molecular identification and characterization of turkey IFN-γ gene

The cDNAs of turkey and chicken interferon gamma (IFN-γ) were cloned and the functional activity of turkey and chicken IFN-γ was compared. The coding region of turkey IFN-γ gene encodes a predicted mature protein of 145 amino acids with a molecular weight at 16.8 kDa. Compared with type I IFN, the IFN-γ between turkey and chicken also had the same size and high degree of identity at the nucleotide (96.0%) and amino acid (96.4%) sequence. Turkey IFN-γ was cross-reactive with chicken cells. Both turkey and chicken IFN-γ could induce production of nitric oxide by turkey or chicken macrophages. Turkey IFN-γ also had similar degree of sensitivity to heat and pH 2.0 as chicken IFN-γ. The functional activity of both turkey and chicken IFN-γ could be neutralized by a monoclonal antibody specific to chicken IFN-γ to a similar extent. These results indicated that IFN-γ protein was cross-reactive between turkey and chicken.     

Monitoring the earliest amyloid-beta oligomers via quantized photobleaching of dye-labeled peptides

Misfolding and aggregation of amyloid-β peptide (Aβ) are widely recognized as causative events in Alzheimer’s disease (AD). Contrary to earlier hypotheses, recent studies have identified soluble Aβ oligomers as the pathogenic agents and documented neurodegenerative effects from species as small as dimers and trimers. As such, detection and characterization of the earliest Aβ oligomers are paramount to understanding, preventing, and treating AD. We have exploited quantized photobleaching from individual dye-labeled Aβ peptides to characterize monomers and small oligomers tethered to the surface of functionalized coverslips. In this way, we have directly determined reproducible distributions of peptide species in various sample environments. Fresh samples (30 pM peptide) at pH 7.4 consist primarily of monomers and dimers. Both acidic conditions (pH 5.8) and zinc coordination promote greater association, which is further increased in samples prepared from more concentrated stocks. Acid- or zinc-promoted association is largely prevented and/or reversed by addition of the β-sheet breaker peptide iAβ5 or the chelator clioquinol, respectively. These results are qualitatively consistent with bulk-solution studies of Aβ40 and demonstrate a powerful approach for definitively characterizing Aβ oligomers. The methodology utilized here holds promise for assessing aggregation promoters and inhibitors and investigating peptide association in its earliest stages.     

Structure of the extracellular hemoglobin of Biomphalaria glabrata

The hemoglobin of Biomphalaria glabrata was purified to homogeneity by gel filtration column followed by anion exchange chromatography. The dissociation products were analyzed by a 5–15% gradient polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS-PAGE) giving a band of 270 kDa and a band of 180 kDa after reduction with β-mercaptoethanol. The same profile was obtained in a 3.5% agarose gel electrophoresis containing SDS (SDS-AGE) but showed additional bands of higher molecular weight. These bands were proposed to be monomers, dimers and trimers, since they showed a good correlation in a plot of R_f versus log M_r. After partial reduction in a two-dimensional SDS-AGE, the proposed monomers and dimers produced two and four bands, respectively, likely indicating one to four chains crosslinked by disulfide bridges. Digestion with four different proteases yielded several equivalent fragments with molecular weights multiples of its minimum molecular weight (17.7 kDa). The circular dichroism spectrum of the protein showed a characteristic high α-helix content (70%). It was proposed that this hemoglobin is a pentamer with a molecular weight of aproximately 1.8×10³ kDa, assembled by five 360-kDa subunits, each formed by two 180-kDa chains linked in pairs by disulfide bridges and each of these chains, in turn, comprised by ten heme binding domains linked in tandem. These data are compared to the published information for other planorbid extracellular hemoglobins.     

Diverse Metastable Structures Formed by Small Oligomers of α-Synuclein Probed by Force Spectroscopy

Oligomeric aggregates are widely suspected as toxic agents in diseases caused by protein aggregation, yet they remain poorly characterized, partly because they are challenging to isolate from a heterogeneous mixture of species. We developed an assay for characterizing structure, stability, and kinetics of individual oligomers at high resolution and sensitivity using single-molecule force spectroscopy, and applied it to observe the formation of transient structured aggregates within single oligomers of α-synuclein, an intrinsically-disordered protein linked to Parkinson’s disease. Measurements of the molecular extension as the proteins unfolded under tension in optical tweezers revealed that even small oligomers could form numerous metastable structures, with a surprisingly broad range of sizes. Comparing the structures formed in monomers, dimers and tetramers, we found that the average mechanical stability increased with oligomer size. Most structures formed within a minute, with size-dependent rates. These results provide a new window onto the complex α-synuclein aggregation landscape, characterizing the microscopic structural heterogeneity and kinetics of different pathways.     

Dimeric and trimeric antibodies: high avidity scFvs for cancer targeting

Recombinant antibody fragments can be engineered to assemble into stable multimeric oligomers of high binding avidity and specificity to a wide range of target antigens and haptens. This review describes the design and expression of diabodies (dimers), triabodies (trimers) and tetrabodies (tetramers). In particular we discuss the role of linker length between V-domains and the orientation of the V-domains to direct the formation of either diabodies (60 kDa), triabodies (90 kDa) or tetrabodies (120 kDa), and how the size, flexibility and valency of each molecules is suited to different applications for in vivo imaging and therapy. Single chain Fv antibody fragments joined by polypeptide linkers of at least 12 residues irrespective of V-domains orientation predominantly form monomers with varying amounts of dimer and higher molecular mass oligomers in equilibrium. A scFv molecule with a linker of 3-12 residues cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody, approximately 60 kDa). Reducing the linker length below three residues can force scFv association into trimers (triabodies, approximately 90 kDa) or tetramers ( approximately 120 kDa) depending on linker length, composition and V-domain orientation. A particular advantage for tumour targeting is that molecules of 60-100 kDa have increased tumour penetration and fast clearance rates compared with the parent Ig (150 kDa). We highlight a number of cancer-targeting scFv diabodies that have undergone successful pre-clinical trials for in vivo stability and efficacy. We also briefly review the design of multi-specific Fv modules suited to cross-link two or more different target antigens. Bi-specific diabodies formed by association of different scFv molecules have been designed as cross-linking reagents for T-cell recruitment into tumours (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics). The more challenging trispecific multimers (triabodies) remain to be described.     

Tracking Mutant Huntingtin Aggregation Kinetics in Cells Reveals Three Major Populations That Include an Invariant Oligomer Pool

Huntington disease is caused by expanded polyglutamine sequences in huntingtin, which procures its aggregation into intracellular inclusion bodies (IBs). Aggregate intermediates, such as soluble oligomers, are predicted to be toxic to cells, yet because of a lack of quantitative methods, the kinetics of aggregation in cells remains poorly understood. We used sedimentation velocity analysis to define and compare the heterogeneity and flux of purified huntingtin with huntingtin expressed in mammalian cells under non-denaturing conditions. Non-pathogenic huntingtin remained as hydrodynamically elongated monomers in vitro and in cells. Purified polyglutamine-expanded pathogenic huntingtin formed elongated monomers (2.4 S) that evolved into a heterogeneous aggregate population of increasing size over time (100–6,000 S). However, in cells, mutant huntingtin formed three major populations: monomers (2.3 S), oligomers (mode s_20,w = 140 S) and IBs (mode s_20,w = 320,000 S). Strikingly, the oligomers did not change in size heterogeneity or in their proportion of total huntingtin over 3 days despite continued monomer conversion to IBs, suggesting that oligomers are rate-limiting intermediates to IB formation. We also determined how a chaperone known to modulate huntingtin toxicity, Hsc70, influences in-cell huntingtin partitioning. Hsc70 decreased the pool of 140 S oligomers but increased the overall flux of monomers to IBs, suggesting that Hsc70 reduces toxicity by facilitating transfer of oligomers into IBs. Together, our data suggest that huntingtin aggregation is streamlined in cells and is consistent with the 140 S oligomers, which remain invariant over time, as a constant source of toxicity to cells irrespective of total load of insoluble aggregates.     

Crystallization of dimers of the manganese-stabilizing protein of Photosystem II

The manganese-stabilizing protein (MSP) of Photosystem II was purified from spinach photosynthetic membranes. The MSP was crystallized in the presence of calcium. Despite the apparent purity of the isolated protein, the crystals grew to only about 0.05 mm in their largest dimension. The MSP was analyzed to identify possible sources of protein heterogeneity that could hinder crystal growth. Tandem reverse-phase HPLC/ electronspray ionization mass spectrometry analysis of the MSP showed a major peak and four smaller peaks. All five peaks had molecular masses of 26 535, as expected for mature MSP, indicating the absence of heterogeneities due to covalent modifications. MALDI mass spectroscopy was utilized to identify heterogeneities in the MSP oligomeric state. These measurements showed that purified MSP in solution is a mixture of monomers and dimers, while solubilized MSP crystals contained only dimers. Size-exclusion chromatography and dynamic light scattering were used to probe the effect of the crystallization conditions on the MSP. Size-exclusion chromatography of concentrated MSP showed the presence of aggregates and monomers, while dilute MSP contained monomers. Dynamic light scattering experiments in the absence, or in the presence of 10–50 mM or 100 mM calcium, yielded calculated molecular mass values of 34 kDa, 48 kDa and 68 kDa, respectively. These changes in the observed molecular mass of the MSP could have been caused by the formation of dimers and higher oligomers and/or significant conformational changes. Based on the results reported in this study, a model is presented which details the effect of oligomeric heterogeneity on the inhibition of MSP crystal growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular Ageing of Alpha- and Beta-Synucleins: Protein Damage and Repair Mechanisms

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[³H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated ³H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of ³H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.     

β-Hairpin-Mediated Formation of Structurally Distinct Multimers of Neurotoxic Prion Peptides

Protein misfolding disorders are associated with conformational changes in specific proteins, leading to the formation of potentially neurotoxic amyloid fibrils. During pathogenesis of prion disease, the prion protein misfolds into β-sheet rich, protease-resistant isoforms. A key, hydrophobic domain within the prion protein, comprising residues 109–122, recapitulates many properties of the full protein, such as helix-to-sheet structural transition, formation of fibrils and cytotoxicity of the misfolded isoform. Using all-atom, molecular simulations, it is demonstrated that the monomeric 109–122 peptide has a preference for α-helical conformations, but that this peptide can also form β-hairpin structures resulting from turns around specific glycine residues of the peptide. Altering a single amino acid within the 109–122 peptide (A₁₁₇V, associated with familial prion disease) increases the prevalence of β-hairpin formation and these observations are replicated in a longer peptide, comprising residues 106–126. Multi-molecule simulations of aggregation yield different assemblies of peptide molecules composed of conformationally-distinct monomer units. Small molecular assemblies, consistent with oligomers, comprise peptide monomers in a β-hairpin-like conformation and in many simulations appear to exist only transiently. Conversely, larger assemblies are comprised of extended peptides in predominately antiparallel β-sheets and are stable relative to the length of the simulations. These larger assemblies are consistent with amyloid fibrils, show cross-β structure and can form through elongation of monomer units within pre-existing oligomers. In some simulations, assemblies containing both β-hairpin and linear peptides are evident. Thus, in this work oligomers are on pathway to fibril formation and a preference for β-hairpin structure should enhance oligomer formation whilst inhibiting maturation into fibrils. These simulations provide an important new atomic-level model for the formation of oligomers and fibrils of the prion protein and suggest that stabilization of β-hairpin structure may enhance cellular toxicity by altering the balance between oligomeric and fibrillar protein assemblies.     

Interactions and aggregation of apoferritin molecules in solution: effects of added electrolytes

We have studied the structure of the protein species and the protein-protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of Na(+) and Cd(2+) ions. We used chromatographic, and static and dynamic light scattering techniques, and atomic force microscopy (AFM). Size-exclusion chromatography was used to isolate these two protein fractions. The sizes and shapes of the monomers and dimers were determined by dynamic light scattering and AFM. Although the monomer is an apparent sphere with a diameter corresponding to previous x-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was applied to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The results for the monomers indicate that Na(+) ions cause strong intermolecular repulsion even at concentrations higher than 0.15 M, contrary to the predictions of the commonly applied Derjaguin-Landau-Verwey-Overbeek theory. We argue that the reason for such behavior is hydration force due to the formation of a water shell around the protein molecules with the help of the sodium ions. The addition of even small amounts of Cd(2+) changes the repulsive interactions to attractive but does not lead to oligomer formation, at least at the protein concentrations used. Thus, the two ions provide examples of strong specificity of their interactions with the protein molecules. In solutions of the apoferritin dimer, the molecules attract even in the presence of Na(+) only, indicating a change in the surface of the apoferritin molecule. In view of the strong repulsion between the monomers, this indicates that the dimers and higher oligomers form only after partial denaturation of some of the apoferritin monomers. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization and other phase transitions in the protein solution.     

Structural and functional analysis of native peroxiredoxin 2 in human red blood cells

Peroxiredoxin 2, a typical 2-Cys peroxiredoxin, is the third most abundant protein in erythrocytes. It is understood that the physiologically functional state of peroxiredoxin 2 is the monomer, and that its role in scavenging low levels of H(2)O(2) results in the formation of disulfide-linked dimers, which are reversibly reduced to monomers by the thioredoxin-thioredoxin reductase system. Additionally, peroxiredoxins are highly susceptible to sulfinic acid formation through reactions with various peroxides. This overoxidized form, which is thought to convert peroxiredoxins into molecular chaperones and to be accompanied by a transition to polymeric forms, can be reversed by sulfiredoxins. However, physiological conformational changes and the antioxidant role of erythrocyte peroxiredoxin 2 are still unclear because there is low sulfiredoxin and thioredoxin-thioredoxin reductase activity in erythrocytes. In this study, we examined the structural and redox states of peroxiredoxin 2 in fresh hemolysates and estimated the activities of native and overoxidized peroxiredoxin 2 purified from red blood cells to clear the physiological roles of peroxiredoxin 2 in erythrocyte. Our findings demonstrate that native peroxiredoxin 2 exists as high molecular weight (>160 kDa) oligomers and that decamers or higher order molecular weight oligomers (260-460 kDa) have peroxidase activity. We further showed that peroxiredoxin 2 oligomers, which were predominantly composed of monomers in the reduced form, exert a chaperone activity equal to that of overoxidized peroxiredoxin 2 polymers. These results provide the novel insight that redox-active peroxiredoxin 2 functions in human red blood cells as high molecular weight oligomers that possess peroxidase and chaperone activities.     

Resveratrol Selectively Remodels Soluble Oligomers and Fibrils of Amyloid Aβ into Off-pathway Conformers

Misfolded proteins associated with diverse aggregation disorders assemble not only into a single toxic conformer but rather into a suite of aggregated conformers with unique biochemical properties and toxicities. To what extent small molecules can target and neutralize specific aggregated conformers is poorly understood. Therefore, we have investigated the capacity of resveratrol to recognize and remodel five conformers (monomers, soluble oligomers, non-toxic oligomers, fibrillar intermediates, and amyloid fibrils) of the Aβ1–42 peptide associated with Alzheimer disease. We find that resveratrol selectively remodels three of these conformers (soluble oligomers, fibrillar intermediates, and amyloid fibrils) into an alternative aggregated species that is non-toxic, high molecular weight, and unstructured. Surprisingly, resveratrol does not remodel non-toxic oligomers or accelerate Aβ monomer aggregation despite that both conformers possess random coil secondary structures indistinguishable from soluble oligomers and significantly different from their β-sheet rich, fibrillar counterparts. We expect that resveratrol and other small molecules with similar conformational specificity will aid in illuminating the conformational epitopes responsible for Aβ-mediated toxicity.     

Purification of human leukocyte interferon A derived from Escherichia coli: An aging process to prevent the formation of its oligomers

Recombinant human leukocyte interferon A (rIFN-αA) was purified from Escherichia coli cells by monoclonal antibody affinity chromatography (Ab) and CM-cellulose column chromatography (CM). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CM eluate was found to contain a large amount of rIFN-αA oligomers, in addition to the monomer, linked by intermolecular disulfide bonds. In contrast, the Ab eluate was found to contain a large amount of rIFN-αA monomers. These results show that the oligomers were formed at the CM step. Free SH groups were detected at a significant level in the Ab eluate, but were detected barely in the CM eluate, indicating that the Ab eluate contained some amount of the unstable monomer with free SH groups. To prevent the formation of the oligomers we established a simple and easily scaled-up process, called an aging process, during which the Ab eluate was incubated at pH 3.3 at 37°C for several days. This process is effective for changing the unstable monomer to the stable monomer and thus preventing the formation of the oligomers.     

Cross-linking of native myosin forms oligomers of myosin heavy chain dimers

Chemical cross-linking of native myosin in 0.6 M NaCl with p-phenylene bis maleimide or glutaraldehyde resulted in rapid formation of myosin heavy chain dimers and their oligomers. Monomers and odd-number oligomers disappeared after the prolonged treatment with these reagents. When denatured myosin was cross-linking, myosin heavy chain monomers and odd-number oligomers remained after the prolonged treatment, although dimers and their even-number oligomers were abundant. For high molecular weight markers, myosin heavy chain oligomers formed from denatured myosin with glutaraldehyde or p-phenylene bis maleimide are recommended.     

Structures of the two 3D domain-swapped RNase A trimers

When concentrated in mildly acidic solutions, bovine pancreatic ribonuclease (RNase A) forms long-lived oligomers including two types of dimer, two types of trimer, and higher oligomers. In previous crystallographic work, we found that the major dimeric component forms by a swapping of the C-terminal beta-strands between the monomers, and that the minor dimeric component forms by swapping the N-terminal alpha-helices of the monomers. On the basis of these structures, we proposed that a linear RNase A trimer can form from a central molecule that simultaneously swaps its N-terminal helix with a second RNase A molecule and its C-terminal strand with a third molecule. Studies by dissociation are consistent with this model for the major trimeric component: the major trimer dissociates into both the major and the minor dimers, as well as monomers. In contrast, the minor trimer component dissociates into the monomer and the major dimer. This suggests that the minor trimer is cyclic, formed from three monomers that swap their C-terminal beta-strands into identical molecules. These conclusions are supported by cross-linking of lysyl residues, showing that the major trimer swaps its N-terminal helix, and the minor trimer does not. We verified by X-ray crystallography the proposed cyclic structure for the minor trimer, with swapping of the C-terminal beta-strands. This study thus expands the variety of domain-swapped oligomers by revealing the first example of a protein that can form both a linear and a cyclic domain-swapped oligomer. These structures permit interpretation of the enzymatic activities of the RNase A oligomers on double-stranded RNA.     

Immobilized active monomers of D-glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscles and their coenzyme-binding properties

Experimental conditions favouring the dissociation of tetrameric rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase into active monomers were elaborated. The urea-induced dissociation of the tetramer was shown to be a stepwise process (in 2 M urea only dimers are formed; an increase in urea concentration up to 3 M causes the splitting of the dimers into monomers). The specific activity of immobilized monomers in the glyceraldehyde-3-phosphate oxidation reaction does not differ from that of the parent immobilized tetrameric form. The tetrameric enzyme molecule binds the coenzyme with a negative cooperativity (the first two NAD+ molecules bind with KD below 0.1 microM; for the third and fourth molecules the dissociation constant was determined to be equal to 5.5 +/- 1.5 microM (50 mM medinal buffer, 10 mM sodium phosphate, pH 8.2). The cooperativity of NAD+ binding is preserved in the immobilized preparation of tetrameric dehydrogenase. The immobilized monomers bind NAD+ with KD of 1.6 +/- 1.0 microM. The experimental results are consistent with the hypothesis according to which the association of catalytically active subunits into a tetramer changes their coenzyme-binding properties in such a way that the first two NAD+ molecules bind more firmly to a tetramer than to a monomer, whereas the third and the fourth NAD+ molecules bind less firmly.     

Intrinsic ligand binding properties of the human and bovine α,-interferon receptors

The Type I interferon receptor (IFN-αR) interacts with all IFN-αs, IFN-β and IFN-ω, and seems to be a multisubunit receptor. To investigate the role of a cloned receptor subunit (IFN-αR1), we have examined the intrinsic ligand binding properties of the bovine and human IFN-αR1 polypeptides expressed in Xenopus laevis oocytes. Albeit with different efficiencies, Xenopus oocytes expressing either the human or bovine IFN-αR1 polypeptide exhibit significant binding and formation of crosslinked complexes with human IFN-αA and IFN-αB. Thus, the IFN-αR1 polypeptide most likely plays a direct role in ligand binding.     

Hemocyanins in spiders, IV[1]. Subunit heterogeneity of Eurypelma (Dugesiella) hemocyanin, and separation of polypeptide chains.

The hemocyanin of the North American tarantula Eurypelma californicum (Dugesiella californica) is dissociated at pH 9.6 into monomers (Mr about 70 000) and dimers (Mr about 140 000), which were separated by gel filtration. The monomer peak was resolved by preparative polyacrylamide gel electrophoresis and yielded 4 protein bands, three of which (1, 3 and 4M) are apparently homogeneous. Band 2 contains two sub-fractions (2I and 2II). The dimer peak contains two dimers (bands 4D and 5). Upon treatment with 5mM cysteine the dimer band 5 is dissociated, yielding only one type of monomer identical with band 3. The other dimer, which was only partially dissociated by 10mM EDTA, is most probably a heterodimer, one component being electrophoretically indistinguishable from band 2II. After treatment of the native hemocyanin with sodium dodecylsulfate and analysis in gradient gel slabs, 6 polypeptide chains were observed (labeled a - f). They correspond to the products of alkaline dissociation as follows: band 1 = e, band 2I = a, band 2II = c, band 3 = f, band 4M = d, band 4D = b plus c, band 5 = f. The molecular weights were determined by dodecylsulfate gel electrophoresis in gradient gels, and by sedimentation equilibrium analysis and found to range between 67 000 and 76 000. The sedimentation coefficients are between 4.4 and 4.7 S for the monomers and 6.6 and 6.7 for the dimers. The isoelectric points range from pH 4.5 to pH 5.4. The findings are discussed with respect to the limitations of molecular weight determination by conventional dodecylsulfate gel electrophoresis, to the structure of the hemocyanin oligomers and to possible biological significance.     

Self-assembly of soluble unlinked and cross-linked fibrin oligomers

Self-assembly of soluble unlinked and cross-linked fibrin oligomers formed from desA-fibrin monomer under the influence of factor XIIIa was studied in the presence of non-denaturing urea concentrations. By methods of elastic and dynamic light scattering combined with analytical ultracentrifugation, desA-fibrin oligomers formed in both the presence and absence of the factor XIIIa were shown to be ensembles consisting of soluble rod-like double-stranded protofibrils with diverse weight and size. Unlinked and cross-linked soluble double-stranded protofibrils can reach the length of 350–450 nm. The structure of soluble covalently-linked protofibrils is stabilized by isopeptide γ-dimers. Electrophoretic data indicate a complete absence of isopeptide bonds between α-chains of desA-fibrin molecules. The molecular mechanism of formation of soluble rod-like fibrin structures and specific features of its covalent stabilization under the influence of factor XIIIa are discussed.